Mammalian peroxidases have a role in the metabolism of drugs and toxic chemicals, but to date there has been very little interest shown in this one-electron, oxidative metabolism. Although both hydrogen peroxide and hydroperoxides are found in vivo and peroxidases have a very high turnoover number and are generally nonspecific, little work has been published in cells or in vivo. The primary reason for the lack of apparent peroxidase metabolism is that the free radical metabolites are reduced by biochemical reducing agents regenerating the parent substrate. As a consequence of this reduction, free radicals of ascorbate, glutathione, and NAD(P)H may be formed. The radical metabolites of the latter two species GS and NAD(P) react with oxygen to form a variety of oxygen-derived species known as reactive oxygen species. We have recently carefully compared the oxidation of these biochemical reducing agents by the tyrosyl radical in a peroxidase system. In addition, we have begun an investigation of the mechanisms of dichlorofluorescein peroxidase oxidation. The oxidation of 2-7-dichlorofluorescin (DCFH) in cells to the highly fluorescent product 2-7-dichlorofluorescein (DCF) has become a popular assay for oxidative stress with nearly 400 publications using this assay. The mechaniam of this oxidation is poorly understood and will be the subject of investigation. From the literature, the oxidation of 2-7-dichlorofluorescin is known to be dependent on a strong oxidant such as hydroxyl radical, peroxynitrite, or compund I of a peroxidase. We believe the peroxidase-dependent oxidation of DCFH forms a free radical intermediate, which reacts with oxygen to form superoxide. If this assay for oxidative stress creates reactive oxygen species in cellular systems, it will be of limited utility. Using ESR, oxygen consumption, and fluorescence, we will investigate the mechanisms of DCFH oxidation by cells and a model peroxidase.